Immunohistochemical expression of angiotensin‐converting enzyme 2 in superficial and deep maxillofacial tissues: A cross‐sectional study

Abstract Background and Aims The involvement of maxillofacial tissues in SARS‐CoV‐2 infections ranges from mild dysgeusia to life‐threatening tissue necrosis, as seen in SARS‐CoV‐2‐associated mucormycosis. Angiotensin‐converting enzyme 2 (ACE2) which functions as a receptor for SARS‐CoV‐2 was reported in the epithelial surfaces of the oral and nasal cavities; however, a complete understanding of the expression patterns in deep oral and maxillofacial tissues is still lacking. Methods The immunohistochemical expression of ACE2 was analyzed in 95 specimens from maxillofacial tissues and 10 specimens of pulmonary alveolar tissue using a semiquantitative immunohistochemical scoring procedure, taking into account all superficial and deep maxillofacial tissue cells. We also explored the associations of age, gender, and anatomical site with expression scores. Results ACE2 was detected in keratinized epithelia (57.34%), non‐keratinized epithelia (46.51%), nasal respiratory epithelial cells (73.35%), pulmonary alveolar cells (82.54%), fibroblasts (63.69%), vascular endothelial cells (58.43%), mucous acinar cells (59.88%), serous acinar cells (79.49%), salivary duct cells (86.26%) skeletal muscle fibers (71.01%), neuron support cells (94.25%), and bone marrow cells (72.65%). Age and gender did not affect the expression levels significantly in epithelial cells (p = 0.76, and p = 0.7 respectively); however, identical cells expressed different protein levels depending on the site from which the specimens were obtained. For example, dorsal tongue epithelia expressed significantly lower ACE2 scores than alveolar epithelia (p < 0.001). A positive correlation was found between ACE2 expression in fibroblasts and epithelial cells (r = 0.378, p = 0.001), and between vascular endothelial and epithelial cells (r = 0.395, p = 0.001). Conclusion ACE2 is expressed by epithelial cells and subepithelial tissues including fibroblasts, vascular endothelia, skeletal muscles, peripheral nerves, and bone marrow. No correlation was detected between ACE2 expression and patient age or sex while the epithelial expression scores were correlated with stromal scores.


| INTRODUCTION
Angiotensin-converting enzyme 2 (ACE2) is a cell surface metallopeptidase that was discovered back in 2000 by two independent groups. It is a regulator of the renin-angiotensin system (RAS) that cleaves angiotensin I and angiotensin II to Angiotensin (1-9) and angiotensin (1-7), respectively. 1,2 In addition to its physiological functions, ACE2 also serves as a functional receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 3 The role of ACE2 in the pathogenesis of severe acute respiratory distress syndrome (SARS) drew the attention of researchers to its expression in human tissues. Quantitative Reverse Transcriptase Polymerase Chain Reaction (QRT-PCR) revealed that ACE2 mRNA is expressed in 72 different human tissues. Immunohistochemical studies offer the advantage of providing visual information about the spatial distribution of ACE2 on the cell surface. 4 ACE2 protein expression was reported in the testis, kidney, heart, lung, bronchioles, nasal mucosa, skin, and vascular endothelium. 5,6 In the oral cavity, ACE2 was detected in epithelial surfaces and in the salivary glands [7][8][9] ; however, ACE2 expression in the majority of deeper oral and maxillofacial tissues such as fibrous tissues, blood vessels, vascular smooth muscles, skeletal muscles, nerves, adipose tissue, bone, and bone marrow is still not thoroughly explored, and many previous immunohistochemical studies were limited by relatively small sample sizes. [7][8][9][10] Recent reports suggested that airborne and fecal-oral routes, as well as direct contact and fomites are possible means of SARS-CoV-2 transmission. 11,12 The oral and nasal tissues are potential targets for the viral infection with symptoms ranging from mild gustatory or olfactory dysfunction to a life-threatening tissue necrosis known as SARS-CoV-2 associated mucormycosis. 13,14 The differences in ACE2 expression may be implicated in the severity of infections, and a number of factors such as age, gender, and ethnicity, are believed to affect the expression of ACE2. 15 Studying the patterns of ACE2 expression in oral and nasal tissues is essential to identify the tissues that could be involved in viral transmission, tissues that are susceptible to damage, and tissues that act as reservoirs for the virus. Identifying the factors that affect ACE2 levels is an essential step in understanding the maxillofacial manifestations of SARS-CoV-2 infections, and may help identify individuals who are more susceptible to severe infections and tissue necrosis. To the best of our knowledge, this is the first study that explores ACE2 expression in all major cell types that constitute the oral and maxillofacial tissues, including superficial tissues, deep soft tissues, bone, as well as pulmonary epithelium. Specimens were randomly obtained from nine locations that represent the maxillofacial and pulmonary alveolar tissues, namely, keratinized gingival and palatal tissues, non-keratinized buccal and labial tissues, ventral tongue, lateral tongue, dorsal tongue, major salivary glands, minor salivary glands, nasal cavity, and pulmonary alveoli. Only the non-pathological biopsy margins were considered for the analysis.
Hematoxylin and eosin (H&E) slides were prepared and examined by a qualified pathologist to verify the suitability of each specimen for inclusion in the study. The exclusion criteria included history of systemic disease or smoking/alcohol consumption (as indicated by the attached clinical reports), malignant and benign neoplastic tissues, tissues with inflammatory infiltrates, tissues with focal or total necrosis, and poorly preserved/small specimens. Patient age and sex were retrieved from the attached clinical reports. A total of 95 maxillofacial specimens and 10 pulmonary alveolar specimens met our inclusion criteria. Patients were classified according to age into young (<18 years), middle-aged (18-49 years), and patients aged (>49 years).
The study protocol was approved by the ethical committee of the College of Dentistry/Baghdad University (Reference number 301721). Negative control slides were prepared by eliminating the primary antibody to exclude the possibility of nonspecific binding.
The positive cell proportion score was calculated as 0 (0%), 1 (<10%), 2 (10%-35%), 3 (35%-70%), and 4 (>70%). Expression scoring was performed by multiplying the staining intensity score by the positive cell proportion score to calculate the staining index (SI). The SI was calculated separately for each cell type, for example, the SI of epithelial cells was calculated independently of that of fibroblasts and vice versa. A score of 4 or less was regarded as weak expression while a score of 6 or more indicated strong expression. The scoring procedure was blindly performed by two qualified investigators, and the SI values were averaged for further comparative evaluation as described previously. 16

| Statistical analysis
For groups in which scores were normally distributed, the two-tailed t-test was used to compare the means of two groups. Oneway analysis of variance (ANOVA) was used to compare multiple groups. For groups in which scores were not normally distributed,

Mann-Whitney U and Kruskal-Wallis tests were used instead. The
Chi-square test was used to compare ACE2 expression levels between the different age groups, and between males and females.
Pearson's correlation coefficient was used to analyze the relationship between stromal and epithelial ACE2 expression. A p value of less than 0.05 was considered statistically significant. All statistical analyses were carried out using IBM SPSS statistics 26 software.

| RESULTS
The mean age of the patients was 38.58 years, 54 patients were males and 51 were females. ACE2 expression in epithelial and stromal cells was not significantly different between males and females (p = 0.69).
Patients of different age also showed no significant difference in ACE2  Table 1.  Table 2.
The nasal respiratory epithelia expressed ACE2 in 73.35% of the cells while pulmonary alveoli expressed ACE2 in (82.54%) of the cells.
No significant difference in ACE2 expression was detected between the nasal and pulmonary epithelia (p > 0.99).
ACE2 staining was detected in fibroblasts (63.69%), and it was consistent across different oral and respiratory tissues. Vascular endothelia expressed ACE2 in 58.43%, of the cells, and the expression was lower in the blood vessels below the dorsal epithelium of the tongue compared to gingival mucosa (p = 0.002).
Blood vessels below non-keratinized epithelium also expressed lower endothelial ACE2 scores than those from the keratinized gingiva (p = 0.01). The median epithelial and endothelial ACE2 expression scores are presented in Figure 1. In salivary glands, ACE2 was expressed in mucous acinar cells

| DISCUSSION
Since the nasal and oral cavities are both implicated in SARS-CoV-2 infections, this study was carried out to provide a comprehensive understanding of ACE2 expression giving consideration to the wide variety of cells that constitute the maxillofacial tissues. We also sought to identify the factors that affect ACE2 levels in maxillofacial and pulmonary alveolar tissues.
In this study, the relationship between ACE2 expression and patient age and gender was explored. Although ACE2 SI values did show interindividual variation, there were no statistically significant differences between males and females or between younger and older patients. Some studies contradict this result. A review by Getachew and Tizabi in 2021 suggested that ACE2 declines with age, especially in males. 15 One immunohistochemical study reported higher ACE2 levels in the oral epithelial cells of patients older than 49 years. 10 Multiple studies, however, agree that males do not express significantly different ACE2 levels compared with females, and that ACE2 expression does not seem to be correlated with the patient's age. [16][17][18][19] This study agrees with a previous immunohistochemical and RNA sequencing study that reported high ACE2 expression in respiratory epithelia. 20 However, One RNA sequencing study contradicted this result suggesting that the expression level in the respiratory epithelium was low, and that olfactory epithelium was comparatively more markedly positive for ACE2. 21 However, RNA studies usually have the limitation that they cannot measure the protein contents of the tissue directly. 4 One noticeable feature of ACE2 expression was that identical cells expressed different ACE2 levels depending on the sites from which the tissue specimens were obtained. For example, the keratinized mucosa of the dorsal surface of the tongue expressed lower ACE2 levels than the keratinized mucosa of the gingiva.
Previous studies that performed public bulk RNA sequencing data set analysis, 22 single-cell sequence data set analysis, and immunohistochemistry 9 detected different epithelial ACE2 levels in different oral sites; however, they reported that ACE2 levels in the tongue were higher than in other oral sites. 9,22 The fact that these studies did not subdivide the tongue mucosa into dorsal, lateral, and ventral may ACE2. It is likely that ACE2 performs other physiological functions besides its role in the renin-angiotensin system. 7 This might explain the variability in its levels in different organs.
Some reports implicate SARS-CoV-2 infection in causing oral manifestations such as dysgeusia, salivary dysfunction, and oral mucosal lesions. 13 The high ACE2 signals detected in our study are in agreement with previous studies, 9,22  The detection of high levels of ACE2 in the salivary ducts and acinar cells is in agreement with previous immunostaining studies, 7,8 and supports the conjecture that salivary glands may act as reservoirs for the virus. 28 The expression was consistent in major and minor salivary glands, and both serous and mucous acini expressed comparable levels of ACE2. One immunohistochemical study detected ACE2 in salivary ducts but not in salivary acini. 29 The small sample size in their study and the fact that they did not use HIER technique may be the reason behind this discrepancy.
Recently One remarkable finding of this study was that epithelial ACE2 expression scores were positively correlated with stromal ACE2 scores.
This was also true for samples from the nasal cavity. To the best of our knowledge, this finding was not reported previously in the literature;

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
This study was approved by the ethical committee of the College of Dentistry at the University of Baghdad (Reference number 301721).

TRANSPARENCY STATEMENT
Noor Allawi affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.